Enzyme I: [MND,AG] The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(II)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(II)) promotes essentially complete dimerization of EI(H189A) at pH 7.5 and 20 C, 5 mM pyruvate (with 2 mM Mg(II)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. With 2 mM Mg(II) at 15-25 C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with an apparent association constant of 1.0E6 /M. The binding of PEP to EI(H189A) is synergistic with that of Mg(II). Thus, physiological concentrations of PEP and Mg2+ increase, whereas pyruvate and Mg2+ decrease the amount of dimeric, active, dephospho-enzyme I. Short C-terminal rod of myosin II from Acanthamoeba: (EF, AG, EK, FM) The Acanthamoeba heavy chain myosin II C-terminal fragment (dimer Mr 34,920) being studied contains residues 1373-1509 of the long dimeric coiled-coil rod studied earlier in our laboratory and possesses a part of the a-helical coiled-coil structure and the C terminal unstructured 29 residue tail harboring the 3 phosphorylation sites (Ser residues) which have been mutated to either Asp or Ala for our studies The 153 residue peptide used has 2 Trp residues engineered into a loop structure between the 14 residue flag peptide and the N-terminal of the C-terminal rod fragment. Wild type, Ala and Asp mutants at the 3 phosphorylation sites are being compared in order to study the role of these positions in stabilizing the coiled-coil structure. High salt (600 mM KCl) in 20 mM K-phosphate at pH 7.5 favors the monomeric rod of myosin II and these are the conditions used in the present studies. Human NKx-2.5 homeodomain protein stability and DNA interactions:(EF, AG, JM, JF) The NKx-2.5 homeodomain (residues 1-80) is homologous with the vnd/NK-2 homeodomain protein from Drosophila melanogaster previously studied in our laboratory by Gonzales et al. 2001, Biochemistry 40, 4923-4931). We have used the C56S mutant of the wild-type NKx-2.5 homeodomain since preliminary studies showed that Cys56 caused problems both in the isolation and subsequent thermal unfolding studies. Thermal unfolding studies using DSC and CD measuremnets as well as ITC measurements to determine the thermodynamics of the interaction of NKx.2.5 (C56S) with specific 18 bp DNA are being performed. CARMIL from Acanthamoeba and heterodimer Capping Protein Interaction: (MND, AG, KR, JH) Purified CARMIL (subunit Mr = 121610) preparations in the absence of Capping Protein are homogeneous as evidenced in sedimentation velocity experiments which show a single symmetrical boundary. At 9.4 micromolar subunit, CARMIL has a sedimentation coefficient of 7.1 S, under which conditions the protein is >90% dimer. This indicates that the CARMIL dimer is quite asymmetric with a frictional coefficient of ca. 2.0 or about 2-fold greater than that of a spherical particle. Global fitting of sedimentation equilibrium data at 4 C obtained at 34 and 36 h at 7500 rpm for two CARMIL preparations gave excellent fits to a monomer - dimer equilibrium model with an association constant of 6.0E6 /(M monomer) at pH 7.0. Once CARMIL and the Capping protein (CP) had been separately purified and the extinction coefficients determined, these proteins were mixed in a known ratio to give 1.7-fold CP:CARMIL for a sedimentation equilibrium experiment. After reaching equilibrium, the data have been analyzed Only one model was consistent with the fact that CARMIL monomer could not be detected and that the presence of CP promotes dimerization of CARMIL. For the mixture, interference data are fitted well to a model with free CP (calculated from [3.28 micromolar total - complexed CP]), 13-15% CARMIL dimer, and 85-87% CARMIL dimer complexed to one equivalent of CP. Moreover, the calculated association constant, log K = 6.4 (/M) for CP binding to CARMIL dimer from the sedimentation equilibrium data is in agreement with the value measured for the affinity of CP for CARMIL in a BioCore assay. Human ClpP and ClpX Chaperone Interaction: (MND, AG, MRM, SGK) In both sedimentation velocity and sedimentation equilibrium experiments, purified human ClpP (hClpP) was shown to be a monodisperse heptamer of 24,166 Mr subunits (7.0 S). This result was surprising since in E. Coli, the two 7-membered rings of ClpP are assembled under native conditions into a stable tetradecamer without free heptamer present. Since in the mitochondria hClpP is complexed to hClpX, an approach to equilibrium at low speeds was used (with either absorption or intrference optics at 4-12 C) to examine mixtures of hexameric hClpX with heptameric hClpP.in the presence of ATPgammaS. It has been found that the interaction of hexameric ClpX with heptameric ClpP strongly stabilizes the tetradecameric structure of ClpP in a complex flanked by hexameric rings of ClpX (analogous to the E. Coli ClpAP complex; Maurizi e al., 1998, Biochemistry 37, 7778-7786). Calmodulin-like protein: (MND, AG, AP, PR) We were approached to conduct sedimentation equilibrium experiments to detect a monomer-dimer equilibrium, since the elution position of Dr. Reddy's cloned calmodulin-like protein from gel filtration columns indicated a larger size than expected for a monomeric protein. Sedimentation equilibrium studies of this protein showed no evidence of association of monomers (4962 Da) in the absence or presence of 1 mM Ca(II) at either 4 or 25 C. Cel 45 mutant (cellulase) and Cel 45 core: (MND,AG, GR) We also were approached by G. Rialdi to run sedimentation velocity experiments at both pH 4.2 and pH 7.8 of an inactive mutant D10N of Cel-45 core (containing no carbohdrate) with Mr 22,865 and Cel-45 wild-type cellulase, containing 8-12 k Da carbohydrate (Mr 40,200 ). Cel-45 appeared to form a dimer at the higher pH in isoelectric electroforesis studies. The core Cel-45 was monomeric at both acid and neutral pH, as anticipated. However, the wild-type Cel-45 in sedimetnation velocity runs also was monomeric at both pH's. Prof. Rialdi now is investigating whether Cel-45 interacts with ampholytes in gels at the higher pH.